348 



thernioreKulator and theriiiometer, the enzyine was added and the temperature kept 

 constant. 



After a few hours the tubes were taken out, alcalised and the indoxyl oxidised 

 hy strong shaking, then acidified, by which a very fine, equally divided, purely blue 

 precipitate of indigo-blue is obtained, which allows colorimetrically to establish the 

 intensity of action with sufficiënt exactness. 



it proved wholly indifferent whether in these experiments use was made of the 

 indican of Indigofera or of Polygonum. Evidently it is the same in both plants. 



Great attention should however be paid to the degree of acidity of the indican 

 Solutions. The most favourable enzyme action was observed at the rate of about 0.5 cc. 

 normal acid per 100 cc. liquid. An increase of the acid to 2 cc. slackens the reaction 

 notably; likewise the addition of alkali to feebly alkaline. Acid salts, as kalium- 

 bioxalate and kaliumbiphosphate, act in the same way as free acids. 



The quantities of the enzymes employed for the experiments amounted to 2 — 60 

 milligrams of finely powdered crude enzyme per 10 cc. of the 34 pCt. indican-solution. 



First of all was now established the maximum temperature at which the action 

 of the enzymes ceases entirely, that is, where the enzymes are nearly suddenly 

 destroyed. For Indigofera this maximum is at 75° C, but when using a great deal 

 more enzyme a feeUle action could still be observed at 78° C. which however quite 

 ceased at 80" C. For Polygonum the maximum temperature is at 55" C, and in large 

 quantities a feeble action was still observable at öo^C. For this determination thetubes 

 were placed, for Indigofera, at /2''.^, 75», and 80» C, for Polygonum. at 520.$, 55", 

 58" and 60" C. For both enzymes the action at the rising af the temperature diminishes 

 very quickly near the maximum. In a similar way were found as maximum tempera- 

 tures for Sacchammyces sphaericus about 60° C. for Phajns grandiflorus 67", and 

 for emulsine 70" C. 



After this the optimum tcmpcratures lor the enzyme action were fixed, for 

 Indigofera by searching between 55° and 65" the ma.ximum intensity of indigo-form- 

 ation, testing all temperatures from 55", 57", 59" C. and so an. The strongest in- 

 version was found at 61" C. both for powdered crude enzyme and for enzyme-solution 

 in lopCt. common salt and lopCt. calcium chloride. Changes in the degree of 

 alkalinity or acidity wifliin the narrow confines between which enzyme action is at 

 all possible, deplace the optimum temperature but little M. A difference in tempera- 

 ture of i" C. was only to be observed between 61" and 62"; at 62" C. the decompo- 

 sition was certainly a little feel)ler, but between 60" and 61" C. there existed some 

 doubt. At lower temperatures liistinct differences in the intensity of the decompo- 

 sition could only be noted at intervals of 2" C. 



The enzyme of Polygonum. cxaniuied in the same way between 35" and 45" C, 

 gives the most copious production of indigo at 42" C, with a rapiil decrease in action 

 aliove, a slow onc below that point. 



') Mr. van D c 1 d c n fuiiiid iii)on addition of acid lioth for IndiKofcr,! and i'otygonum 

 a risins of 1° in optinuiin, which however disappeared whcn tho employed Solutions 

 of crude indican were diluted with an equal volume ot water and tluii, lieforc the 

 aiUlition of enzyme. were brought to the same acidity. 



